Even though the protein should be denatured by all that urea, we find such gels sometimes look nicer if stop the reaction with SDS and protease K, and/or phenol extract the remains of the protein before loading the high-urea gel.
++++++++++++++++++++++++++++++++++++++++++ Phoebe A. Rice Dept. of Biochemistry & Molecular Biology The University of Chicago pr...@uchicago.edu ________________________________________ From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Opher Gileadi [opher.gile...@sgc.ox.ac.uk] Sent: Saturday, September 30, 2017 3:44 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Off topic: denaturing urea gels In addition to the previous suggestions: With very small gels, the sample composition and depth (in the well) have a strong effect on the resolution. Rinse the wells with TBE buffer just before loading, as urea from the gel diffuses into the well and may prevent the sample from settling at the bottom. Minimize the amount of salt in the samples; try to load very small volumes (1-3 ul), even if this means longer exposures later.
